DESCRIPTION: (Adapted from the application abstract) Neisseria gonorrhoeae is one of the two major pathogens involved in the majority of cases of bacterial sexually transmitted genital infection and pelvic inflammatory disease (PID). We have shown that the forms of C3 bound to N. gonorrhoeae at the cervical level in vivo correlate with measurements of these components in vitro and with postulated mechanisms of the resultant clinical syndromes. We have recently found that N. gonorrhoeae modulates interactions with C3 by directly binding the alternative complement pathway regulatory protein factor H to sialylated lipooligosaccharide (LOS) and to loop 5 of porin protein (Por) IA. The classical complement pathway regulator C4 binding protein (C4BP) also appears to directly bind N. gonorrhoeae Por, similar to Streptococcus pyogenes M protein, which binds both factor H and C4BP. This proposal will define the molecular mechanisms of these interactions. In the first Specific Aim, we propose to further define the molecule(s) on the surface of a serum resistant PorA strain (15253) of N. gonorrhoeae that mediate factor H binding, along with the specific regions of factor H that are involved in these binding interactions. We will first create isogenic strains of 15253 by moving the Por IB molecule from strain MS11 (which does not bind factor H) into strain 15253. If factor H binding is completely abrogated, hybrid Por mutants will be created to define the region of Por responsible for binding. If not, we will attempt to define a second gonococcal factor H binding site by TnMax-71 mutagenesis. Regions in factor H that bind to Por will be identified using recombinant factor H (rH) deletion mutant proteins' generated in Chinese hamster ovary (CHO) cell lines. Construction of these rH mutant proteins is integral to Specific Aim 2, which seeks to define the role of glycosylation of factor H in binding to N. gonorrhoeae. We have observed that strain FA19, which expresses PorIA but differs by one amino acid at position 181 of exposed loop 5 from strain 15253, binds human factor H but does not bind non-glycosylated rH produced in a baculovirus system. We will express fully glycosylated deletion mutant rH proteins in CHO cells using overlap extension PCR and compare binding to non-glycosylated baculovirus rH. Site directed mutagenesis of position 181 of PorIA in strain 15253 will also be done to examine whether rH binding can be regained. In Specific Aim 3, we will construct mutants by shuttle mutagenesis using strains FA19 (PorIA) and F62 (PorIB) (the latter does not bind C4BP and is genetically competent) to define the gonococcal region responsible for C4BP binding. A confirmatory approach to define the loop necessary for C4BP binding will be to use synthetic peptides encompassing the putative exposed regions of the different porin loops to inhibit C4BP binding to N. gonorrhoeae in flow cytometry. Functional correlates will also be demonstrated in bactericidal assays. Finally, Specific Aim 4 will examine a set of clinical isolates that lack the 3F11 lactosamine sialylation site yet can be sialylated and, further, appear to endogenously sialylate their LOS. We have cloned the sialyltransferase from one of these isolates and will insertionally inactivate the gene. The resultant strains will be characterized for: i) the ability to sialylate (endogenously or in the presence of exogenous CMP-NANA), ii) their serum sensitivity in bactericidal assays, and iii) their LOS structure.